The Expression Of Hypoxia Inducible Factor-1α And Vascular Endothelial Growth Factor In Arteriosclerosis Obliterans Patients' Ischemia Lower Limbs.

Objective: To investigate the expression of hypoxia inducible factor-1αand its related gene in arteriosclerosis obliterans patients'ischemia lower limbs, and to provide the theoretical basis of HIF-1αgene therapy for ischemic disease.Materials and Methods:Materials and Methods11.1 Getting specimen15 patients with lower limbs ischemic disease were selected from June 2005 to June 2006 in vascular surgery department of our hospital.All the 15 patients were performed amputation and we choosed the bole vessels and ischemic muscular tissue of their amputated limbs as the specimen and were compared to the normal tissue. Among them there were 10 males, 5 females.The age is 62-85 years old, and the average is 78 years old.Diagnostic criteria: patients went to hospital because of severe pain of their legs and feet and their toes became darken. The Fontaine artery stages wereⅣ. We found obliteration or severe stenosis of anterior tibial artery, posterior tibial artery and fibular artery and also found segment obliteration or severe stenosis or thrombogenesis of popliteal artery and femoral artery by vascular ultrasound examination. Some cases were confirmed by angiography. All cases were performed up-knee joint amputation.The specimens were the tissues from six following position:①superficial femoral artery②anterior tibial artery③posterior tibial artery④blood vessels of ischemic muscular tissue of legs⑤ischemic muscular tissue of legs⑥muscular tissue around the necrosis tissue of feet.1.2 Immunohistochemistry analysis of the expression of HIF-1αand vascular endothelium growth factor (VEGF) and microvessel density (MVD)The specimens were fixed through 10% formaldehydes and deaquated and then undergone paraffin imbedding and performed 3-4μm thickness serial sections and SP method immunohistochemistry dyeing.We buy the HIF-1α, VEGF and CD34 polyclonal antibody from Boster company in Wuhan. The working concentration was 1:100, and the CD34 was the mode that could be used immediately. The PBS fluid was used to be negative controlacts.1.3 Assessment standard of the outcomeWe considered as the positive HIF-1αprotein outcome if we found brown and yellow grains in cell nucleus and endochylema, negtive if non- response. We choosed eyesight under the low power lens and then used Cooled CCD to collect pictures under the 200×eyesight and IPP analysis software to determine the expression gray scale value.We also considered as the positive CD34 protein expression outcome if we found brown and yellow grains in vascular endothelial cell endochylema.The determine method of MVD: we choosed 5 high micro-vascular position under 100×light microscope and counted blood vessels under 200×, single colorated cell was also counted.The situation was exclused if the numbers of vascular endothelial cell were higher than 8.Materials and Methods2 2.1 Ischemic limbs model establishment of rabbitsWe selected 15 white New Zealand rabbits as laboratory animals. The sex of rabbits was unlimited and the average weight was 3 kg. The rabbits were bought from animal laboratory of foundation medicine college, Jilin University.Experimental methods: Model building: we used Sumianxin 0.2-0.3ml/kg to give intramuscular injection which was producted in military veterinarian institute of military medical sciences academy in Changchun. The rabbits were backlying and fixed and then degermed. We paved aseptic surgical drapes and cut open the median skin from crural ligament to knee joint of the left upper legs to the femoral artery. We exposed the femoral artery completely with the deep femoral artery, lateral circumflex femoral artery, pudendal artery, popliteal artery and arteria saphena. We cut off the crural ligament, ligated theexternal iliac artery,pudendal artery above the crural ligament level and the deep femoral artery, the lateral circumflex femoral artery and the superficial epigastric artery below the crural ligament level, and ligated the popliteal artery and arteria saphena from distal end. So the femoral artery was rejected completely from crural ligament level to popliteal artery and arteria saphena. All the animals were given intravenous transfusion with 0.9% normal saline (50ml) and antibiotic (Cefradine, 40mg/kg) during operation.2.2 Observation methods2.2.1 Common clinical assessmentThe clinical manifestation was assessed according to before operation, 1 day after operation, 7 days after operation and 14 days after operation in each animal which contained the observation of activity, color, ulcer, gangrane and amotic situation of the affected limbs.2.2.2 The color Dopplar examination of blood flow and angiography before and after operation.The color Dopplar examination: we used ALOKA5500 and the frequency of the detecting head was 10MHz.Angiography: we performed angiography examination before operation,1 day and 14 days after operation, and selected 3 rabbits randomly every time. The animals were given routine anesthesia before angiography.We cut open the gastro-skin of the cervical part to expose the left carotid artery. If it needed to be performed the secondly angiography, its right carotid artery should be exposed. We used 18G transfixion pin to puncture the carotid artery and deliver forward 3 cm to aorta crotch, and then injeced 3-5ml contrast material (Ultravist 300) with hands and took photography continuely at the same time. The results were dealed with the digital subtraction radiography method.2.2.3 Getting specimen and immunohistochemistry analysisWe obtained the tissues from the gastrocnemius muscle of affected extremity for histology study at 5 minutes, 2 hours, 10 hours, 24 hours, 48 hours, 7 days, 14 days after operation and the tissues from opposite side as the comparison.2.3 The methods of immunohistochemistry analysis and outcome assessment were as same as 1.2 and 1.3.3. Statistical treatment methodAll statistical results were analysised by statistical software-Instat, and we used t-test to group compare.ResultsMethod 11.1 The expression of HIF-1αproteinHIF-1αis appeared to be brown and yellow grain and mainly expressed in the nucleus and endochylema of blood vessel wall cells (including endothelial cell, smooth muscle cell and fibrocyte).No expression was found in normal skeletal muscle tissues.We found the increased HIF-1αin ischemic muscle tissues and bole vessel wall cells of ischemic limbs of ASO patients.The highest expression of HIF-1αwas found in posterior tibial artery and the difference was obvious (p<0.001).The increased expression in blood vessel wall cells was related to the ischemic level.More severe ischemia, more increased expression. However, we didn't found obvious difference in different ischemic muscle tissues. For example, the muscle tissue around the necrosis tissue of foot was more ischemic than the leg, but the expression of HIF-1αhad no difference. Compared with the ischemic bole vessel wall cells, the expression of HIF-1αin muscle tissue tent to be low level.1.2 The expression of VEGF proteinNo expression was found in normal skeletal muscle tissues. The VEGF is mainly expressed in the endochylema of blood vessel endothelial cells.It's also positive expressed in smooth muscle cells.The highest expression of VEGF was found in posterior tibial artery and the difference between posterior tibial artery and superficial femoral artery, blood vessels of ischemic muscular tissue of legs, ischemic muscular tissue of legs, muscular tissue around the necrosis tissue of feet, was obvious (p<0.05). Although there was no difference between posterior tibial artery and anterior tibial artery, the expression in anterior tibial artery was lower than posterior tibial artery. The increase of VEGF was consisted with the expression of HIF-1αin ischemic bole vessel endothelial cells and ischemic muscular tissues.We didn't found obvious difference of VEGF expression in different ischemic tissues. Compared with the ischemic bole vessel wall cells, the expression in muscle tissue tent to be low level.1.3 The counts of MVD CD34 protein is appeared to be brown and yellow and mainly expressed in the endochylema of blood vessel endothelial cells. And it's also expressed in the neogenetic micro-vessels of ischemic muscle tissues and vessel wall cells. We found the most numbers of neogenetic vessels in posterior tibial artery and least in ischemic muscular tissue of legs.The numbers of new vessels in superficial femoral artery, anterior tibial artery, blood vessels of ischemic muscular tissue of legs, ischemic muscular tissue of legs and muscular tissue around the necrosis tissue of feet were obviously less than in posterior tibial artery through statistical analysis (p<0.001).Method 22.1 Clinical evaluation:Lameness, pallor and low skin temperature were found in all 15 animals, and different degrees of cutex necrosis in distal end of left lower extremity (knee and ankle) were found in one animal at 7 days after operation. Another one got wound infection.2.2 Color Dopplar examination: We found the blood flows were well engorged in arteria iliaca, femoral artery and popliteal artery before operation and asteria in muscle tissues. Arteria iliaca, femoral artery and popliteal artery were not found after operation and the asteria blood flows were disappeared in muscle tissues.2.3 Angiography: We performed angiography before operation, 1 day after operation and 14 days after operation.Compared with the opposite side, we found the absence of femoral artery and no establishment of compensatory circulation 1 day after operation. The images we obtained obviously displayed slowly engorged blood flows and compensatory circulations built by microvessels from upper legs.2.4 Histology study: We found that the right lower extremity was normal skeletal muscle tissue when we examed the sections by microscope.The atrophy, regeneration and fibrosis of muscle fiber and necrotic muscle tissue among the normal tissue were found obviously in muscular sections of left lower extremity 24 hours after operation. We used immunohistochemistry method to analysis the expression of HIF-1α, VEGF and MVD. The time points of expression of HIF-1α, VEGF at 5 minutes, 2 hours, 10 hours, 24 hours, 48 hours, 7 days and 14 days refer to the table 2. The expression of HIF-1αwent to peak at 2 hours, and depressed from 24 hours to 48 hours, and nearly tent to be normal level after that. The expression of VEGF went to peak at 10 hours, and depressed to be nearly normal level at 48 hours. The expression of CD34 was not found at any time point.DiscussionHypoxia inducible factor 1(HIF-1) is the transcription factor generally existed in vivo of mammalian and humen beings under the anoxia condition. HIF-1 combines the target gene to creat the accommodative reaction to hypoxia and ischemia by adjusting the transcription and the regulation after transcription. HIF-1 controls the composition of more than 20 kinds of angiogenesis factors, for example the VEGF, EPO and ET-1 et al. HIF-1 contains two subunits,α-subunit andβ-subunit and mainly exists in heterodimers form. HIF-1 can not be found under normoxia condition because of rapid degradation. However HIF-1βand the mRNA of two subunits express generally in different tissues and cells and we found HIF-1βin all the cells. HIF-1αincreased significantly because the degradation was interfered when hypoxia happened, and HIF-1αprotein transfer to nucleus and combined with HIF-1βto be dimeride complex-HIF-1, participating the transcription regulation of hypoxia reaction genes. HIF-1αis the particular subunit of HIF-1 regulated by hypoxia.HIF-1αpromotes the expression of many kinds of angiogenesis factor including VEGF and the physiological and pathologic angiogenesis and to form lots of microvessels in blood vessel wall. Because of no effective collateral circulation probablely due to the relatively insufficient expression of HIF-1α,it seems to be infernal cycle that the condition of ischemia and hypoxia is aggravated. That's maybe the originated basis of ASO. And this kind of microvessel hyperplasia mainly happens in great vessels which explains the reasons why the great vessels are usually involved in ASO patients. Compared with the superficial femoral artery, there is obvious ischemia and hypoxia in distal end of limbs. So the highest expression of HIF-1αwas found in posterior tibial artery which is the bole artery in the distal end of limbs. And the microvessels in muscle tissue of legs can be supplied through compensatory circulation, so the expression of HIF-1αis not as obvious as posterior tibial artery and the difference is significant (p<0.001). Although the ischemia in muscular tissue around the necrosis tissue of feet is worse than ischemic muscular tissue of legs with the ischemia in skeletal muscle, there is no increased expression of HIF-1α(p>0.05). Because the expression of HIF-1αin muscle tissues is lower than that in blood vessel wall cells, it's difficult to form the compensatory circulation which lead to the ischemia and necrosis of the distal end of limbs.VEGF is mainly expressed in the endochylema of blood vessel endothelial cells. It's also positive expressed in smooth muscle cells. The increasing of VEGF in bole blood vessel endothelial cells and ischemic muscle tissues is as same as the expression of HIF-1α, which improves the physiological and pathologic angiogenesis and the development of angiosclerosis.CD34 protein is appeared to be brown and yellow and mainly expressed in the endochylema of blood vessel endothelial cells. MVD is usually considered one of the index to reflect the angiogenesis. CD34 marked blood vessel endothelial cells can add up the new MVD correctly and is the monitoring index reflecting the expression of HIF-1αand VEGF.The expression of HIF-1αwent to peak at 2 hours, and depressed from 24 hours to 48 hours, and nearly tent to be normal level after that in ischemic muscle tissues of rabbits. The expression of VEGF went to peak at 10 hours and stop increasing, but higher than normal tissues, and depressed to be nearly normal level at 48 hours and still higher than normal tissues.The expression of HIF-1αand VEGF increases uniformly (P<0.01).And the experimental results indicate that high expression of HIF-1αand VEGF was found in acute ischemic muscle tissues and the significant depression in chronic ischemic muscle tissues which is still higher than normal tissues.Therefore, the research of ASO patients and animal model indicates that the insufficient expression of HIF-1αand VEGF in chronic ischemic skeletal muscle tissues will be the theory basis of remedial angiogenesis therapy.Researchers studied the expression of HIF-1αin ischemic myocardiac tissue, brain tissue, pulmonary tissue and tumor tissue, which was limited to analyse the expression of HIF-1αin acute ischemic animal model and human skeletal muscle tissue.But we never found the reports about the analysis of the expression of HIF-1αin chronic ischemic diseases which this study will lay particular emphasis on.Peripheral arterial occlusive disease (PHOD) tent to be a high incidence rate (12%), and often damage the microvessels diffusely at advanced stage of disease which lead to severe ischemia in region tissue. Anticoagulant therapy can not improve the ischemic condition of the limbs'ending effectively. So we usually use the artery bypass operation and angioplasty. However many patients can not be performed the operation for the anatomy distribution and damage degree of vessels, and 20%-30% patients faced to be amputated even performing surgical and interventional operation.Remedial angio-regeneration can improve the angiogenesis in ischemic tissue which offer a wide space in treating this kind of disease.It's the therapy aiming to improve the angiogenesis and blood supply through the direct application of angiogenesis factor, particular transgenic and cell therapy. These years angio-regeneration gene therapy has been applied for the experimental and clinical study of ischemic disease. But peoples pay all attentions to angiogenesis factors including VEGF which produce a marked effect only at the different stage of angiogenesis with side effect or being short of efficacy. HIF-1αpromotes producting many kinds of angiogenesis factors as upstream regulatory factor.So it's important to analyse the expression of HIF-1αand its relative gene in ischemic tissue, and provide the theory directions for remedial angio-regeneration and effective therapy for peripheral arterial occlusive diseases.Conclusion1. The expression of HIF-1αand VEGF in vessels and muscle tissues of ischemic extremity of ASO patients is higher than normal tissue and the difference is obvious.CD34 displays the increasing of MVD.2. The expression of HIF-1αand VEGF in the vessels of ischemic extremity of ASO patients is higher as the worse ischemia and the difference is obvious. CD34 displays the increasing of MVD.3. HIF-1αand VEGF expression were generally increase in acute ischemic muscle,whereas were generally decrease in chronic ischemic muscles. The expression of HIF-1αand VEGF in the muscle tissues of ischemic extremity of ASO patients does not increase as the worse ischemia and tents to decrease relatively compared with the blood vessel wall cells, which may be the theoretic basis of remedial angio-regeneration.